Status - voluntarily withdrawn in 2010

Organizations involved:

Ortho Biotech Inc. – Manuf.; R&D; Tech.; USA mark.

Johnson & Johnson Co. – Parent

Ortho Pharmaceutical, Inc. – Former

Janssen-Kyowa – Japan mark.

Cross ref: See the Monoclonal Antibodies entry above.

Description: Muromonab-CD3 Sterile Solution or Ortho-clone OKT3 is an aqueous formulation of muromonab, a purified murine (mouse) monoclonal antibody, directed against the CD3 (T3) receptor on the surface of human T-cells (T-lymphocytes) cultured using the murine ascites method. Muromonab is 93% monomeric immune globulin G type 2a (IgG2a), composed of two heavy chains of about 50 kDa each and two light chains of about 25 kDa each in the standard double-chain Y configuration of human immunoglobulin molecules (antibodies). Muromonab-CD3 is manufactured by the classic Kohler and Milstein and ascites culture methods for murine Mab production invol-ving generation of a hybridoma cell line and its in vivo culture in the ascitic fluid/peritoneal sac of mice. The antibody has a total molecular weight of ~150 kDa.

Orthoclone OKT3 was the first monoclonal antibody product approved for in vivo use. Orthoclone OKT3 reverses graft (transplant) rejection by blocking the function and reducing the production of cytotoxic T lymphocytes or T-cells (CTLs) responsible for the tissue inflammation and destruction that occur during acute transplant rejection. Orthoclone OKT3 does not react with or affect hematopoietic or other human cells or tissues.

Orthoclone OKT3 is packaged in 5 mL ampules, each containing 5 mg (1 mg/mL) of muromonab-CD3 in a phosphate buffer (pH of about 7.0) containing monobasic sodium phosphate (2.25 mg), dibasic sodium phosphate (9.0 mg), sodium chloride (43 mg), and polysorbate 80 (1.0 mg) in Water for Injection. The product is stored at 2-8˚C (refrigerated).

Nomenclature: CD3 Mab [BIO]; Orthoclone OKT 3 [TR]; Muromonab-CD3 [FDA USAN INN JAN]; murine monoclonal antibody for Cluster of Differentiation 3 antigen [SY]; OKT-3 [SY]; anti-CD3 [SY]; T3 monoclonal antibody [SY]; NDC 59676-101-01 [NDC]

The company reports no CAS RN (according to Materials Safety Data Sheet, Dec. 1987).

Biological.: In vitro cytolytic assays show that mumo-monab-CD3 binds to CD3 on T-cells and blocks both the generation and function of these immune effector cells. Mumo-monab-CD3 reverses graft rejection by blocking the function and reducing the production of cytotoxic T-cells (CTLs) which are responsible for the tissue inflammation and destruction that occur during acute transplant rejection. Mumo-monab-CD3 does not react with hematopoietic cells or tissues. Mumo-monab-CD3 is active in vitro at concentrations of 1,000 ng (1 µg)/mL. In early kidney rejection trials, mean trough levels of mumo-monab-CD3 rose during the first 1-3 days of treatment and then averaged 900 ng/mL on days 3-14.

The target of muromonab-CD3, the CD3 receptor, is 20 kDa epsilon chain glycoprotein occurring only on the surface of mature T-cells and medullary thymocytes. The CD3 designation identifies the specificity of the antibody as Cell Differentiation (CD) cluster 3, as defined by the First International Workshop on Human Leukocyte Differentiation Antigens. The binding of muromonab-CD3 to the T-cell CD3 receptor complex interferes with cell-mediated immune responses, particularly cytotoxic T lymphocyte (CTL) responses, blocking the functional activity of T-cells involved in recognition of foreign antigens, including antigens involved in acute allograft rejection. The T-cell CD3 receptor is associated in vitro with the antigen recognition structure of T-cells and is essential for signal transduction in cytolytic assays. Binding of muromonab to T-cells results in early activation of the T-cells, leading to cytokine release, followed by blockage of lymphocyte function (which returns to normal within one week of termination of treatment). Both the generation and function of immune effector cells are blocked.

At the time of the product’s approval, no experimental model for the activity of muromonab-CD3 was available (the antibody is highly species-specific), so few, if any, animal studies were performed. The CD3 antigen recognized by muromonab-CD3 is only present in humans, chimpanzees, and gorillas (unavailable for testing during development of the product). Also, the fine molecular structure was unknown.

Companies.: Orthoclone OKT3 was developed and is manufactured by Ortho Pharmaceutical Inc./Ortho Biotech Inc., a subsidiary of Johnson & Johnson (J&J), now CBER/FDA est. no. 1610. Ortho-clone OKT3 is marketed in the U.S. by Ortho Biotech Inc. The product is marketed in most countries worldwide by J&J affiliated companies. It is marketed in Japan by Janssen-Kyowa, a joint venture of Kyowa Hakko and Janssen, a J&J affiliate.

Muromonab-CD3 was voluntarily withdrawn from the market due to decreased utilization. OKT3 supplies are expected to be exhausted by mid-2010.

Manufacture: Muromonab-CD3 is produced by the now classic Kohler and Milstein method for murine ascites antibody production using a murine hybridoma cell line. The original hybridoma was produced by immunizing mice with human peripheral T lymphocytes (T-cells) bearing the CD3 receptor antigen, fusing antibody-producing B-cells from the spleens of mice with murine myeloma cells in the presence of propylene glycol, and selecting a resultant CD3-specific monoclonal antibody-secreting hybridoma. Such fused hybridoma cells essentially contain the full genotype from both source cells. The hybridoma cells can secrete the immunoglobulin (monoclonal antibody) that was produced by the immunized human donor B-cell, while the myeloma genome confers “immortality” or the ability to replicate and be cultured in established cell lines. The selected hybridoma cell line produces an antibody uniquely reactive with the CD3 (T3) antigen on human T lymphocytes. The primary hybridoma seed stock has been shown to be free of murine viruses.

The hybridoma is cultured in vivo in pathogen-free male CAF3 mice. Mice are handled in special areas by gloved, gowned, and masked personnel. Mice are inoculated with either hybridoma cells grown from secondary seed stock or with ascites fluid from another previously inoculated mouse. After receiving an intraperitoneal injection of hybridoma cells, the cells are incubated/cultured within the peritoneal cavity or gut of the animal. Ascitic fluid containing the hybridoma-expressed monoclonal antibody is harvested from the peritoneal cavity, processed by centrifugation, and stored frozen. At the time of approval, production involved using 1,000 CAF3 mice every other week (with mice held in quarantine for one week). The expressed muromonab-CD3 is tested for monoclonal antibody concentration by gel chromatography and for identity by isoelectric focusing.

The crude monoclonal antibody is purified by a combination of ammonium sulfate fractionation and anion exchange chromatography. The purified Mab is tested to ensure identity, purity, potency, molecular integrity, and the absence of murine viruses, mycoplasma, and polynucleotides. The purified antibody is diluted to obtain the final bulk product, which is sterilized by membrane filtration. The final container product is formulated as a sterile solution in glass ampules.

The final container product is tested prior to release for identity (isoelectric focusing compared to standard), purity, potency (compared to an OKT3 standard), molecular integrity (SDS/PAGE patterns); HPLC (ion-exchange and gel permeation), sterility, safety (no extraneous toxic contaminants), and pyrogenicity. Microorganism (virological) testing includes testing: Mouse Antibody Production; Mouse Thymic Virus Isolation; LCM Virus Isolation by Intracerebral Inoculation; Epizootic Diarrhea of Infant Mouse; Diagnosis of Lactic Dehydrogenase Elevating Virus in Test Articles by Mouse Inoculation; Mouse Cytomegalovirus; Xenotropic Murine Leukemia Virus; and Ectopic Murine Leukemia Virus. All lots must be assayed to be virus-free.

FDA class: Biologic PLA

CBER class: Blood And Blood Derivatives

CBER to CDER: Among the products transferred within FDA on June 30, 2003

Approvals: Date = 19860619, first approval, PLA 84-149/ELA 84-150, granted to Ortho Pharmaceutical (est. no. 0996), subsidiary of Johnson & Johnson; Indication = for the treatment of acute allograft rejection in renal transplant recipients

Date = 19920914; Indication = licenses revoked and granted (reissued) to Ortho Biotech, Inc. (est. no. 1157)

Date = 19930608; PLA supplement; Indication = for treatment of heart or liver transplant rejection resistant to steroid therapy

Indications: [full text of "INDICATIONS AND USAGE” section of product insert/labeling]:

ORTHOCLONE OKT3 is indicated for the treatment of acute allograft rejection in renal transplant patients.

ORTHOCLONE OKT3 is indicated for the treatment of steroid-resistant acute allograft rejection in cardiac and hepatic transplant patients.

The dosage of other immunosuppressive agents used in conjunction with ORTHOCLONE OKT3 should be reduced to the lowest level compatible with an effective therapeutic response. (See WARNINGS and ADVERSE EVENTS: infections, Neoplasia; DOSAGE AND ADMINISTRATION).

Status: Product was withdrawn from the market due to low sales.

In Europe, this was the first phamaceutical to be approved under the directive 87/22/EWG, a precursor of the European Medicines Agency (EMEA) centralized approval system in the European Union.

Tech. transfer: Exemplary U.S. patents covering aspects of Orthoclone OKT3 include 4,361,549, Drs. P.C. Kung and G. Goldstein. This patent describes the OKT3 monoclonal antibody expressed by a hybridoma (patent deposit, ATTC CRL 8001) from the fusion of splenocytes from immunized CAF1 mice with P3X63Ag8Ul myeloma cells and its uses. Based on time in FDA regulatory review (under 35 USC §156), the expiration date for this patent was extended 209 days, to June 18, 2000.

Trials: Ethical barriers prevented clinical testing of injection of Orthoclone OKT3 in normal human volunteers for formal pharmacokinetic studies. Safety and efficacy were demonstrated in human trials in transplant recipients, with relatively little preclinical efficacy-related animal testing (as discussed in the Biological section).

In all patients studied, a rapid and concomitant decrease in the number of circulating CD3+, CD4+ and CD8+ T-cells was observed within minutes after administration. Between days 2 and 7, increasing numbers of circulating CD4+ and CD8+ T-cells are generally observed. CD3+ cells are not detectable in treated patients due to modulation of CD3 on cells by muromonab-CD3. Changes in the CD4+ and CD8+ cells have little or no impact on graft rejection. CD3+ T-cells reappear rapidly and reach pretreatment levels within one week of halting therapy. This reappearance of CD3+ T-cells is attributed to the development of neutralizing antibodies to muro-monab-CD3, which block its ability to bind to the CD3 receptor at low concentrations.

Medical: A typical dosage regimen for adult allograft recipients is 5 mg (one vial) administered daily as a single bolus intravenous injection for 10-14 days. Regimens are available for pediatric patients. Muromonab-CD3 is only used in conjunction other immunosuppressive therapeutics, such as steroids and antimetabolites, for the treatment of kidney and liver trans-plant rejection.

A rapid and concomitant decrease in the number of circulating CD3+ T-cells (and also cells that are CD2+, CD4+, or CD8+) has been observed within minutes of administration of OKT3 to patients. The binding of muromonab-CD3 to the CD3 receptors on T-cells leads to the release of numerous cytokines and lymphokines (which can lead to adverse events, such as Cytokine Release Syndrome).

Market The 2007 Average Wholesale Price (AWP) is $1,142.62 (Red Book, 2007).

Ortho/J&J does not report Orthoclone OKT3 annual sales. However, sales are likely in the range of several $100 million.